1. Field of the Invention
The present invention relates to compositions and methods for fusion protein separation.
2. Related Art
Expression systems utilizing fusion proteins are a well-accepted technology for the production of recombinant proteins. In such systems, the fusion partner facilitates the expression and purification of the desired protein. The fusion partner is frequently used to provide a “tag” which can facilitate the subsequent purification of the fusion protein. However, in order to recover the desired protein in its native form or in a pharmaceutically acceptable form, the fusion partner must be removed once the fusion protein is isolated. The most widely used method to remove the fusion partner involves the use of specific cleavage enzymes such as thrombin, factor Xa or enterokinase (Wassenberg et al., Protein Sci. 6:1718 (1997); Schlumpberger et al., Protein Sci. 9:440 (2000); Zaitseva et al., Protein Sci. 5:1100 (1996)). This involves the insertion of a unique amino acid sequence that is specific for cleavage by the cleavage enzyme between the desired protein and the fusion partner. The desired protein can be recovered by the cleavage of the fusion protein with the cleavage enzyme (e.g., thrombin).
Thrombin is a trypsin-like serine protease which will cleave peptide bonds using the serine amino acid. The specificity of thrombin has been studied by a number of investigators. The previously known thrombin cleavage sites are as follows (Chang, Eur. J. Biochem. 151:217 (1985); GST gene fusion system handbook, Amersham Biosciences, Edition AA, p. 88-89).    1) P4-P3-Pro-Arg/Lys↓P1′-P2′, wherein P3 and P4 are hydrophobic amino acids and P1′ and P2′ are non-acidic amino acids. The Arg/Lys↓P1′ bond is cleaved.Examples:
P4P3ProArg/LysP1′P2′ALeuValProArgGlySerBMetTyrProArgGlyAsnCIleArgProLysLeuLys(SEQ ID NOS:1-3)    2) P2-Arg/Lys↓P1′, wherein either P2 or P1′ is Gly. The Arg/Lys↓P1′ bond is cleaved.Examples:
P2Arg/LysP1′AAlaArgGlyBGlyLysAla
The most frequently used thrombin cleavage sequence is Leu-Val-Pro-Arg-Gly (SEQ ID NO:4) or Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1), which is derived from the sequence in bovine factor XIII (Takagi et al., Biochemistry 13:750 (1974)). Cleavage occurs at the arginine residue, resulting in the protein of interest being extended at its amino-terminal end by either a Gly or Gly-Ser. This thrombin cleavage sequence is also adopted in several commercially available expression plasmids, including the pGEX series (Amersham Biosciences) and the pET series (Novagen).
More recently, the Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) sequence was further modified to include a glycine-rich linker containing the sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5) located immediately before or after the thrombin cleavage site Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) (Guan et al., Anal. Biochem. 192:262 (1991); Hakes et al., Anal. Biochem. 202:293 (1992)).
While cleavage by thrombin in the currently known linker sequence region is reasonably specific, it is not absolute. Although thrombin is a reasonably specific enzyme, it can use a variety of different amino acid sequences as its cleavage site. If the target protein contains thrombin cleavage sites, then the cleavage can occur at those sites, resulting in the production of an internally cleaved protein, rather than the desired full length protein.
For example, when Halobacterium halobium L11 protein, which contains an internal thrombin cleavage sequence, was expressed as a GST fusion protein that contained the Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:1) thrombin cleavage site between the GST tag and L11, treatment with thrombin resulted in cleavage within the target protein L11, and not between L11 and the GST tag (Porse et al., J. Mol. Biol. 276:391 (1998)). The Cdc14p protein of Saccharomyces cerevisiae also has an internal thrombin cleavage sequence. When the Cdc14p protein was expressed as a GST fusion protein containing a Ser-Gly-Gly-Gly-Gly-Gly-Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:6) thrombin cleavage site, the fusion protein was cleaved at the internal site within Cdc14p as well as the site within the thrombin cleavage linker (Taylor et al., J. Biol. Chem. 272:24054 (1997)).
The present invention provides a novel linker sequence for thrombin cleavage which provides superior specificity to those known in the art.